JP2001333796A - Method of wipe examination for microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of examination capable of being carried out without sticking a culture medium component to a subject for examination in the case of acting a fiber layer on the subject for examination to carry out the wipe examination for microorganisms. SOLUTION: This method of wipe examination for microorganisms features carrying out cultivation by putting or pasting the fiber layer by which the surface of the subject for examination is wiped on a culture medium containing a water-soluble polymer compound layer as the upper layer to confirm the presence of the microorganisms.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for collecting and culturing microorganisms. More specifically, the present invention relates to a method for collecting and testing microorganisms in food and the environment by wiping.

[0002]

2. Description of the Related Art Conventionally, in the examination of microorganisms, a predetermined area of an object to be inspected is wiped with a gauze, a cotton swab, or the like, washed with sterilized water or sterilized physiological saline, and adhered cells are sterilized with sterilized water or sterilized physiological saline. A method of culturing microorganisms by suspending in a saline solution and applying the obtained suspension to an agar medium prepared in advance, or after previously dissolving and sterilizing a powder agar medium , Keep it at about 45 ° C,
An aliquot of the agar medium was dispensed into a sterilized Petri dish or the like containing an aliquot of the above suspension, and after pour-out, the agar was solidified and cultured at a constant temperature. Has been performed by a method such as counting. In order to carry out the wiping test for microorganisms more simply and promptly, it is preferable that such a laborious and time-consuming preparation of a medium can be omitted. In addition, since a large amount of plastic petri dishes are used in the usual method, there remains a problem that a large amount of waste is generated after culturing.

[0003] In the republished WO 97/24432,
A microorganism culture medium comprising a porous matrix layer and a water-soluble polymer compound layer is disclosed. When performing an environmental test of microorganisms using this, after adding water or water containing nutrients for culturing microorganisms from the porous matrix layer, the porous matrix layer is brought into direct contact with the test object. By wiping, microorganisms can be collected, and the medium can be used as it is. However, since the test is performed by directly contacting the test object, the medium components and the dissolved water-soluble polymer compound may adhere to the test object, where microorganisms may grow. There has been a demand for the development of means for testing microorganisms.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an inspection method which can be performed without directly attaching a medium component to an inspection object when performing a wiping inspection of microorganisms by directly contacting a fiber layer with the inspection object. It is in.

[0005]

Means for Solving the Problems The inventor of the present invention has made intensive studies to solve the above-mentioned problems, and as a result, has found that the intended purpose can be achieved by adopting the following structure.
The present invention has been achieved. (1) The microbial wiping characterized in that the fiber layer from which the surface of the object to be inspected has been wiped is placed on or adhered to a medium having a water-soluble polymer compound layer as an upper layer and cultured to confirm the presence of the microorganism. Inspection methods. (2) The method for wiping off microorganisms according to (1), wherein the fiber layer is a nylon meltblown nonwoven fabric having a basis weight of 40 to 100 g / m ² . (3) The method for wiping microorganisms according to (1) or (2), wherein the water-soluble polymer compound layer is a polyvinyl alcohol layer containing a medium component.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The method for wiping microorganisms according to the present invention is a method for inspecting the presence of microorganisms.After wiping an object to be inspected with a sterilized fiber layer, a previously prepared water-soluble polymer compound layer is formed on the upper layer. Place the wiped fiber layer on a sterilized medium having cultivation, and perform cultivation to confirm the presence of microorganisms, or attach the wiped fiber layer to a similar medium and perform culturing, This is a method of wiping microorganisms for checking the presence or absence of microorganisms adhering to a test object, confirming the presence.

The medium having an upper layer of a water-soluble polymer compound layer used in the present invention is in a state in which microorganisms can grow by adding water or a nutrient component for culturing the microorganisms. It is necessary to have a water-soluble polymer compound layer on the upper layer. At this time, the water-soluble polymer compound layer may be composed of a single layer or a plurality of layers.

As the medium having an upper layer of a water-soluble polymer compound layer used in the present invention, a medium which is usually sealed in a container in a sterilized state is used, and particularly a medium which is sealed in a sheet-like container. However, since it is lightweight and excellent in portability, it can be preferably used. For example, a sheet-shaped medium in which the medium portion is sealed with a transparent film and an adhesive sheet, a sheet-shaped medium in which a fine foam sheet and a film having a smooth surface are integrated, and the like are mentioned.

The medium component used in the present invention can be used without any particular limitation as long as it is suitable for the microorganism to be detected. For example, nutrient components for culturing microorganisms can be used. Further, an antibiotic such as an antibiotic that suppresses the growth of microorganisms other than for the purpose of detection, an antibiotic such as a synthetic antimicrobial agent, a coloring agent, a surfactant, or a selection agent such as an inorganic salt may be added. Further, a tetrazolim salt such as 2,3,5-triphenyltetrazolium chloride or a PH indicator may be added to make the formed colonies easy to see, and a color-developing or fluorescent enzyme substrate may be added to detect a specific microorganism. May be added.

As nutrients for culturing microorganisms, general-purpose liquid media or components obtained by removing agar from an agar medium can be used. For example, a mixture of yeast extract, peptone, glucose, meat, Extract / peptone mixture, peptone / soybean peptone / dextrose mixture, etc., and dipotassium phosphate and / or sodium chloride added to them, etc. For testing Escherichia coli and coliform bacteria, sodium desoxycholate / peptone / ammonium iron citrate・ Sodium chloride ・ dipotassium phosphate ・ lactose ・ neutral red mixture, peptone ・ lactose ・ dipotassium phosphate ・ eosin Y ・ methylene blue mixture etc., for staphylococcus, meat extract ・ peptone ・ sodium chloride ・ mannitol ・ phenol red・ Egg yolk mixture, peptone, meat extract, yeast Kiss pyruvate sodium glycine lithium chloride-tellurite liquid egg yolk mixture and the like,
For Vibrio, yeast extract, peptone, sucrose, sodium thiosulfate, sodium citrate, sodium cholate, ferric citrate, sodium chloride, bovine bile, bromthymol blue, thymol blue mixture, etc. Bovine brain extract, heart extract, peptone, glucose, dipotassium phosphate, sodium nitride, bromthymol blue, 2,3,5-triphenyltetrazolium chloride mixture, etc. For fungi, peptone / glucose mixture, yeast extract / glucose A mixture, a potato extract / glucose mixture and the like are used.

In the medium of the present invention, a resin film may be laminated as a mount under the water-soluble polymer compound layer. This resin film is a film used as a backing paper when producing the water-soluble polymer compound layer. This film may be removed from the water-soluble polymer compound layer if necessary. As a raw material of the resin film, at least one resin selected from polyester, polyolefin, and nylon is used. It is preferable to use a resin film made of these resins, since the adhesiveness to the pressure-sensitive adhesive sheet is improved. In addition, it is preferable that the surface of the resin film be smooth, since the adhesiveness to the fine foam sheet is improved. The medium may be cut into any shape and used.

The fibers forming the fibrous layer used in the present invention include nylon fibers, polyacrylonitrile fibers,
Synthesis of polyvinyl alcohol (PVA) fiber, ethylene vinyl acetate copolymer fiber, polyester fiber (preferably hydrophilic polyester fiber), synthetic polyolefin fiber (preferably polyolefin fiber hydrophilized), polyurethane fiber, etc. Fiber, semi-synthetic fiber such as rayon fiber, wool (animal hair), silk, cotton fiber,
Examples thereof include natural fibers such as cellulose fibers and pulp fibers, and inorganic fibers such as glass fibers. Of the above fibers, more preferred are nylon fibers, cotton fibers, cellulose fibers, and rayon fibers. A fiber layer made of a fabric such as a woven fabric or a non-woven fabric made using these fibers can be used.

As the fiber layer used in the present invention, a nylon melt-blown non-woven fabric produced by a melt-blow manufacturing method or a rayon non-woven fabric produced by a needle punch can be used.
It is preferable because it is hydrophilic and can be easily wiped off. The basis weight of the fiber layer is preferably 40 to 100 g / m ^2, more preferably 50~90g / m ^2, more preferably from 55~80g / m ^2. Weight is 40g
/ M ² , it is difficult to hold the sterilized water added to the fiber layer when wiping is performed, and the fiber layer may be broken at the time of wiping. Conversely, the basis weight is 100 g /
When exceeding m ² significantly, the fiber layer becomes hard, wiping it hardly, when cultured placed on a water-soluble polymer compound layer, the medium component fiber layer contained in the water-soluble polymer compound layer And the growth of microorganisms tends to be delayed. It is necessary to use a sterilized fiber layer.

The water-soluble polymer compound for forming the water-soluble polymer compound layer used in the present invention is preferably 1 × 10 ⁻² Pa · s (viscosity measured at 40 ° C.) after being dissolved in water or the like. Those which exhibit a viscosity of and do not inhibit the growth of microorganisms are preferably used. For example, polyvinyl alcohol, cellulose derivatives, polyacrylic acid derivatives, starch derivatives, proteins, protein derivatives, polysaccharides and the like can be used. Among them, polyvinyl alcohol is more preferable,
The degree of saponification is 75 to 95%, and the molecular weight is 25,000 to 2500.
00 polyvinyl alcohol is particularly preferred.

[0015] The medium used in the present invention comprises at least one medium.
It consists of a water-soluble polymer compound layer. This water-soluble polymer compound layer may be a single layer, or may be a multilayer of two or more layers. When the water-soluble polymer compound layer is a multilayer, it can be prepared by dividing into several layers. When the water-soluble polymer compound layer has a multilayer structure, it is preferable that each layer is in close contact. In addition, even if too many layers are formed, an effect corresponding to an increase in cost due to an increase in the number of manufacturing steps cannot be obtained. Therefore, it is not meaningful to form a multilayer having more than five layers. The basis weight (content of the water-soluble polymer compound itself) in the water-soluble polymer compound layer in contact with the fiber layer is more preferably ² to ²⁰ g / m ^{2, and} still more preferably 3 to 12 g / m ^2. It is.

When the microbial incubator of the present invention is used as a microbial medium, a nutrient component for culturing the microorganism may be added to the water-soluble polymer compound layer before manufacturing the microbial medium. When culturing microorganisms, it is necessary to add water containing nutrients for culturing the microorganisms.

Hereinafter, as an example of a method for producing a sheet medium used in the present invention, an example using a fine foam sheet as a container will be described.

(Preparation of sheet-shaped medium) A fine-foamed sheet composed of a fine foam layer / white polyester sheet is covered with a polypropylene film cover and brought into close contact with each other to obtain a sheet-shaped medium container.

(Preparation of Medium) A solution consisting of a 20% by weight aqueous solution of polyvinyl alcohol, a medium component (including nutrient components for culturing microorganisms), and distilled water was prepared. Finally, a polyvinyl alcohol solution is added so as to be uniform), and this solution is coated on a polyethylene terephthalate mount fixed on a glass plate using an applicator so as to have a proper basis weight, and dried at 110 ° C. for 3 minutes.

(Preparation of a sheet-shaped medium) The prepared medium was cut into a circular shape having a diameter of 5 cm, and the backing side was covered with a fine foam sheet (fine foam layer side) of a sheet-like container with the cover removed.
And cover it again with the foam sheet. Sterilize with ethylene oxide gas, γ-ray, etc. to obtain a sheet medium.

(Usage Method 1) A sterilized nonwoven fabric is used as a fiber layer, and after moistening it with sterilized water, the surface to be inspected is wiped off, a part of the film of the sheet medium is peeled off, and the water-soluble polymer compound is removed. The nonwoven fabric is placed on the top, the film is re-adhered, and the culture is performed at an appropriate temperature.

(Usage Method 2) A sterilized nonwoven fabric is used as a fiber layer, and the sterilized nonwoven fabric is brought into contact with the surface to be inspected as it is, a part of the film of the sheet medium is peeled off, and the nonwoven fabric is placed on a water-soluble polymer compound. Then, sterile water or sterile physiological saline is dropped on the non-woven fabric, the film is re-adhered, and culture is performed at an appropriate temperature.

[0023]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
The measuring method used in the present invention is described below. (1) Weight The weight of a fiber layer cut into a size of 20 cm × 20 cm was measured, and then converted into a weight per 1 m ² (unit: g / g).
m ² ). (2) Weight of Water-Soluble Polymer Compound in Water-Soluble Polymer Compound Layer The water-soluble polymer compound layer cut into a size of 10 cm × 10 cm was dried, and its weight was measured and converted to the weight per 1 m ² . Value (unit: g / m ² ). For the water-soluble polymer compound layer containing the medium component, the value obtained by dividing the medium component by proportional calculation. In the case of a multi-layer structure, the weight was measured after coating and drying each layer, and the difference in each step was determined. (3) Thickness The thickness of the sheet and the film was measured by a techness meter B1 of Toyo Seiki Seisaku-sho, Ltd.

Example 1 A fine foamed sheet having a thickness of 260 μm and a size of 80 m
mx90mm HISTAR-S manufactured by Techno Hirose Co., Ltd.
K (fine foam layer / white polyester sheet)
A cover of a polypropylene film having a thickness of 60 μm and a size of 90 mm × 90 mm was placed on the cover so as to protrude by 10 mm, and was brought into close contact with each other to obtain a sheet-like container. Next, 500 g of a 20% by weight aqueous solution of polyvinyl alcohol,
0.8 g of meat extract, 2.4 g of peptone, 0.8 of NaCl
g, K ₂ HPO ₄ 0.8 g, and add 1 dm with distilled water.
³ , this solution, 400mm × fixed to a glass plate
On a 300 mm × 0.05 mm polyethylene terephthalate mount, it was coated using an applicator so as to be 115 g / m ^2, and dried at 110 ° C. for 3 minutes.
A medium was manufactured. The prepared medium was cut into a circular shape having a diameter of 50 mm, and the backing side was adhered to the central portion of the fine foam sheet from which the cover of the sheet-like container was peeled off, and the cover was covered again and adhered to the foam sheet. This was sterilized with ethylene oxide gas to obtain a sheet medium. From the sheet-like medium, until the entire top surface of the medium is exposed, open the film, use a sterilized polyethylene film-nonwoven fabric laminate as a fiber layer, add 0.5 ml of sterile water to this,
On this nonwoven fabric side, the cutting board was wiped off, and after adding 0.5 ml of water to the nonwoven fabric side of this fibrous layer, the nonwoven fabric side of the fibrous layer was overlaid with the medium, again covered with a film, and sealed.
The cells were cultured in a thermostat at 35 ° C. for 24 hours. The culture produced a red spot. By counting these spots, the number of viable bacteria could be counted.

Example 2 A sheet container produced in the same manner as in Example 1 was used. Also,
As a medium, 500 g of a 20% by weight aqueous solution of polyvinyl alcohol, 2.4 g of meat extract, 7.2 g of peptone, Na
Cl 2.4 g, K ₂ HPO ₄ 2.4 g, 2,3,5-chloride
Triphenyltetrazolium (10 mg) was added, and this was made up to 1 dm ³ with distilled water. This solution was applied to a 400 mm × 300 mm × 0.05 mm polyethylene terephthalate mount fixed on a glass plate using an applicator so as to be 115 g / m ^2. Coated and dried at 110 ° C. for 3 minutes. The medium prepared in this way was used for a 50
mm, and the backing side is adhered to the center of the fine foam sheet from which the cover of the sheet container is peeled off.
The cover was put on again and adhered to the foam sheet. This was sterilized with ethylene oxide gas to obtain a sheet medium. From the sheet-shaped medium, open the film until all the upper surface of the medium is exposed, use a non-woven fabric adhered to a sterilized polypropylene disc as a fiber layer, add 0.5 ml to this, and set the table on the non-woven fabric side. After wiping, the non-woven fabric was peeled off, and the non-woven fabric was overlaid so that the opposite side of the non-woven fabric was in contact with the culture medium. After adding 0.5 ml of sterilized water, the film was covered again, sealed, and sealed in a thermostat at 35 ° C. Cultured for hours. The culture produced a red spot. By counting these spots, the number of viable bacteria could be counted.

[0026]

According to the present invention, the wiping test of the microorganisms in the environment can be easily performed, and the wiping can be performed by the fiber layer containing no medium component, so that the medium component does not adhere to the test object.
Microorganism testing can be performed satisfactorily without any risk of contamination of the test object.

Claims (3)

[Claims] 1. A fiber layer having a surface to be inspected wiped off,
A method for wiping and removing microorganisms, which comprises placing the cells on a medium having a water-soluble polymer compound layer on the upper layer or attaching the cells to the medium and culturing the cells to confirm the presence of microorganisms. 2. The method of claim 1, wherein the fiber layer is a nylon melt-blown nonwoven fabric having a basis weight of 40 to 100 g / m ² . 3. The method for wiping microorganisms according to claim 1, wherein the water-soluble polymer compound layer is a polyvinyl alcohol layer containing a medium component.